Bacterial nitric-oxide synthases operate without a dedicated redox partner

Abstract

Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.

FIGURE 1.

Comparison of mammalian and bacterial NOS structures.

FIGURE 2.

NOS evolution. Phylogenetic tree constructed based on the computational analysis of alignments of NOS-like genes across the whole NCBI data base.

FIGURE 3.

bNOS-dependent NO production in vivo. Effect of nos deletion on total nitrite and nitrate accumulation and growth rate of B. subtilis. WT and Δnos cells were grown in LB medium. Samples were collected every hour and nitrite and nitrate concentration determined in clarified supernatants. Data are shown as mean ± S.E. from three experiments. Circles stand for WT, squares for Δnos mutant, and triangles for ΔnasD,Δnos double mutant cells. Open symbols stand for A₆₀₀ (OD₆₀₀), closed for nitrite and nitrate concentration.

FIGURE 4.

Effect of nos, cysJI, and ykuN deletions on B. subtilis peroxide sensitivity. Strains were grown aerobically in LB to late log phase. An aliquot from each culture was diluted with an equal amount of fresh prewarmed LB for 5 min (diluted). Both diluted and undiluted aliquots were treated with 10 mm H₂O₂ for 30 min; cells were plated on LB agar plates and colony-forming units counted on the next day. Values shown are means and S.D. (error bars) from three independent experiments.

FIGURE 5.

Expression of bNOS in B. subtilis bsNOS strain. Western blot analysis of bNOS expression in B. subtilis WT and bsNOS strains. Cells were grown in LB to A₆₀₀ ∼ 0.5, followed by arabinose and arginine addition. Samples were collected at the indicated time intervals and crude extracts resolved by SDS-PAGE. The membrane was stained by bNOS-specific antibodies.

FIGURE 6.

NO production as a function of bNOS expression in B. subtilis. A, monitoring nitrite and nitrate accumulation in response to bNOS overexpression. B. subtilis WT and bsNOS cells were grown in LB to A₆₀₀ ∼ 0.5, followed by arabinose and arginine addition. Samples were collected every hour and nitrite and nitrate detected in the supernatant. Data are shown as mean ± S.E. from four experiments. B, induction of peroxide resistance by bNOS-mediated NO synthesis. WT and bsNOS strains were grown in LB medium to A₆₀₀ ∼ 0.3, followed by arabinose and arginine addition. After 1 h of incubation, bacteria were challenged with 10 mm H₂O₂ for 30 min. The percentage of surviving cells was determined by colony formation and is shown as the mean ± S.D. from three experiments.

FIGURE 7.

Successful bNOS transplantation from Bacilli to E. coli. A, nitrite and nitrate accumulation in E. coli culture in response to bNOS expression. The plot shows normalized nitrite and nitrate levels in supernatants of E. coli strains harboring pBAD plasmids with WT bNOS from B. anthracis (pNOS_Ban), WT bNOS from B. subtilis (pNOS_Bsu), and mutant bNOS with an increased activity from B. subtilis (pNOS_Bsu(I/V). Experimental conditions are as in Fig. 6A. All values were normalized against the empty vector control. Mean ± S.D. from four experiments. B, induction of E. coli hmp promoter in response to bNOS expression. Experimental conditions are as in A except that all cells contained a pBAD-compatible plasmid expressing a lacZ reporter under the hmp promoter. Induction was calculated based on the Miller unit change. Mean ± S.D. from three experiments. C, representative fluorescent image of bacteria treated with the Cu(II)-based NO-detecting probe (CuFL). Experimental conditions are as in A. Pictures were taken 1 h after CuFL addition. The percent of fluorescent cells (mean ± S.D.) is indicated for each strain.

FIGURE 8.

Effect of peroxide or superoxide on NO production. Monitoring nitrate and nitrite accumulation in response to elevated endogenous peroxide or superoxide level (A) and after H₂O₂ addition to the WT (B) and katA-deficient (C) B. subtilis strains. Strains were grown in LB to A₆₀₀ ∼ 0.3, followed by arabinose addition (time 0) to induce baNOS expression. Hydrogen peroxide was added 2 h later. Samples were collected every half hour; excess of H₂O₂ was removed by treatment with MnO₂ and nitrite and nitrate detected in the supernatant. Open symbols stand for baNOS-expressing strains, closed for the parental strain. Data are shown as mean ± S.E. from three experiments. D, effect of exogenous peroxide on NO production in E. coli. The strain expressing baNOS (pNOS_Ban) was grown in LB to A₆₀₀ ∼ 0.3, followed by arabinose and arginine addition (time 0) to induce baNOS expression. Hydrogen peroxide (1 mm) was added 30 min later. Samples were collected and nitrite and nitrate detected in the supernatant. Data are shown as mean ± S.E. from three experiments.

FIGURE 9.

The oxygenase domain of mammalian NOS is functional in E. coli. Time-dependent nitrite (left) and the sum of nitrite and nitrate (right) formation in E. coli cultures expressing the following proteins: bsNOS, NOS from B. subtilis; nNOS_oxy, oxygenase domain of rat nNOS; nfl, full-length rat nNOS. BL21 stands for control cells that did not express any protein. Data points are the mean of triplicate determinations and are representative of two independent experiments.

FIGURE 10.

Truncated NOSes from simple eukaryotes as an intermediate step in NOS evolution from bacteria. Phylogenetic tree constructed based on the computational analysis of alignments of NOS-like genes. The organisms in which the truncated forms of NOS were detected are marked with asterisks.